Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase: Detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3'-azido-3'-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. Cell extracts incubated with HIV-1 RT and [P-32]ddAMP-terminated DNA primer/template gave rise to P-32-labeled adenosine 2',3'-dideoxyadenosine 5',5"'-P-1,P-4-tetraphosphate (Ap(4)ddA), ddATP, Gp(4)ddA, and Ap(3)ddA, corresponding to the transfer of [P-32]ddAMP to ATP, PPi, GTP, and ADP, respectively. Incubation with [P-32]AZT monophosphate (AZTMP) -terminated primer/template gave rise to the analogous 32P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PPi levels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4(+) or CD8(+) T cells, while PPi was present at 7 to 15 mu M. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4(+) or CD8(+) T cells contained 1.4 to 2.7 mM ATP and 55 to 79 mu M PPi. These cellular PPi concentrations are lower than previously reported; nonetheless, the PPi-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PPi-dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.